There is now widespread interest in the use of fabricated arrays of molecules in the detection and characterisation of analytes. For example, fabricated arrays of polynucleotides are now used widely in DNA sequencing procedures and in hybridisation studies for the detection of genetic variations in a patient.
The fabricated arrays can be designed to include high densities of the same or different molecules immobilised on a solid support surface. This allows the user to generate many results in one experimental procedure. The arrays also have the advantage in that the analytical methods can be automated, thereby allowing a high throughput of samples to be achieved.
The arrays are usually designed with a plurality of individual reactions sites located in spatially-distinct areas on a solid support. In order to produce the arrays in spatially-distinct areas, the most common approach has been through photolithographic techniques. The solid support is coated with a photolabile linker, which only becomes reactive towards a binding ligand following irradiation with light of a suitable wavelength. Spatial resolution is achieved by placing a physical mask on the solid support surface. The pattern of holes in the mask determines the pattern of binding regions on the solid support.
WO-A-95/16204 describes a photolithographic approach using avidin and the photolabile molecule photobiotin. Spatial resolution has also been achieved by passive adsorption. For example, U.S. Pat. No. 5,432,099 discloses binding of the molecules to the solid support surface through a combination of ionic interactions, hydrophobic interactions and Van Der Waals forces.
One particular example of fabricated arrays concerns solid support materials immobilised with nucleic acids. These arrays consist typically of a high-density matrix of polynucleotides immobilised in spatially-distinct regions. Fodor et al Trends in Biotechnology (1994) 12:19-26, describes ways of assembling the nucleic acids using a chemically sensitised glass surface protected by a mask, but exposed at defined areas to allow attachment of suitably modified nucleotide phosphoramidites. Stimpson et al, PNAS (1995) 92:6379-6383, describes the manufacture of fabricated arrays by the technique of “spotting” known polynucleotides onto a solid support at predetermined positions.
In order to maximise the potential and the sample throughput using the array technology, it is essential to fully automate the signal processing from the reaction sites of the arrays. Therefore, it is necessary to perform the imaging of the array, and further mathematical processing of that image, without manual intervention. A difficulty that is experienced with fully automated systems is that each reaction site is sometimes difficult to locate accurately. This may be due to the nature of the array manufacturing process, where it is not possible for the array to be in exactly the same position on each device. The problems may also be due to slight movement of the devices in the apparatus, which may be caused during the washing steps necessary in the assay procedure. As each distinct reaction site may be separated by a distance of only 10-50 μm, it is difficult to ensure that the automated system has accurately positioned each reaction site. For example, movement of the array can be of the same order of magnitude as the distance between each reaction site. This means that predefined windows in the image, for each reaction site to be analysed, cannot be easily used, as the movements may cause the wrong reaction site to fall inside an inspection window which defines the boundary of the reaction site. Alternatively, a reaction site may be missed by the windows, or possibly two sites may fall within the same window. To make processing easier, it is necessary to ensure that only one reaction site falls wholly within each analyte inspection window.
There is therefore a need for improved methods which allow accurate positioning of each discrete reaction site on an array.